Hybridoma cell line that secrets cyproheptadine monoclonal antibodies and preparation method thereof

ABSTRACT

A hybridoma cell line of secreting cyproheptadine monoclonal antibodies with a preservation number of CGMCC No. 14699 belongs to the field of food safety immunological detection. BALB/c mice are immunized through one time immunization with complete freund&#39;s adjuvant, three times of booster immunization with incomplete freund&#39;s adjuvant and one time of rush immunization with cyproheptadine complete antigen without adjuvant; the spleen cells from BALB/C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to cyproheptadine (value of IC50 is 0.37 ng/ml), being suitable for detection of cyproheptadine in food.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority from China Patent Application SerialNumber 201810219839.2, which was filed on Mar. 16, 2018, the entirecontent of which is incorporated herein as reference.

BACKGROUND OF THE INVENTION 1. Technical Field

The invention relates to the hybridoma cell line of secretingcyproheptadine monoclonal antibodies and preparation method thereof,belongs to the technical field of food safety and immunologicaldetection.

2. Background Art

Cyproheptadine (CHP) is an antihistamine and an H1 receptor antagonist,and has the effect of anti-5-hydroxytryptamine, which can inhibit thesatiety center of the hypothalamus to stimulate appetite, increaseweight and promote growth. As a result, many poor manufacturers earnedillegal profits by adding the drug to animal feed and water to quicklygain weight.

However, this illegal behavior can lead to drug residues in animals,which accumulate in large quantities. When people eat these animals,these drugs will accumulate in their bodies. Since cyproheptadine cancause drowsiness, dizziness, weakness, nausea, overeating, weight gain,and even allergic shock, hemolytic anemia, mental disorder, ataxia,thick bronchial secretion, tachycardia, urinary retention and otheradverse reactions, it seriously endangers human health. Therefore, it isurgently need to find a highly specific and sensitive method to detectcyproheptadine residues in food.

The traditional detection methods of cyproheptadine is high performanceliquid chromatography, liquid chromatography tandem mass spectrometry,enzyme linked immunosorbent assay, immune affinity chromatographiccolumn and electrochemical sensor etc. However, these methods ofpretreatment is complicated, time-consuming, and does not apply to alarge number of samples of rapid detection. In order to protect theinterests of consumers, it is necessary to establish an efficient andrapid detection method for cyproheptadine.

Enzyme-linked immunoassay (ELISA) is an extremely efficient, sensitiveand rapid detection methods, which requires less purity and is easy tooperate, being suitable for rapid detection of a large samples. However,the precondition for the detection of cyproheptadine by enzyme-linkedimmunosorbent assay is to obtain monoclonal antibodies with highspecificity and sensitivity to cyproheptadine. Therefore, it is criticalto find a method to prepare monoclonal antibodies with high specificityand sensitivity to cyproheptadine.

The inventors attempted to prepare the monoclonal antibody ofcyproheptadine was prepared by hybrid tumor cells, however it was stillneeded further research and validation that how to preparecyproheptadine haptens and cyproheptadine complete antigens, how to makemice immune, whether the prepared hybrid tumor cell line can secrete themonoclonal antibody of cyproheptadine and What the specificity andsensitivity of the cyproheptadine monoclonal antibody is.

SUMMARY OF THE INVENTION

The purpose of the present invention is to obtain a kind of hybridomacell line of secreting cyproheptadine monoclonal antibodies. Themonoclonal antibody secreted by this hybrid tumor cell line has goodspecificity and detection sensitivity to cyproheptadine (IC50 value is0.37 ng/mL), which could be used to establish the immunologicaldetection method of cyproheptadine, and to detect the residues ofcyproheptadine in food.

The invention provides a hybrid tumor cell line that secretes themonoclonal antibody of cyproheptadine, which has been deposited with thegeneral microbial center of China General Microbial Culture CollectionCommittee (No. 3 yard 1 beichen west road chaoyang district Beijing)under Accession Number CGMCC No. 14697 on Sep. 5, 2017.

The present invention provides a preparation method for a hybrid tumorcell line of secreting cyproheptadine monoclonal antibody, and containsthe following steps:

Step 1: The cyproheptadine hapten and cyproheptadine complete antigenwere prepared. The Cyproheptadine complete antigen were mixed with thesame amount of oil, then the emulsifier was added, and the incompletefreund's adjuvant was obtained after emulsification. The completefreund's adjuvant was obtained by adding mycobacterium into incompletefreund's adjuvant. The oil is paraffin oil or vegetable oil; theemulsifier is lanolin or leaf tween 80; the mycobacterium includes deadseedling.

Step 2: The obtained freund's adjuvant was injected into BALB/c mice forseveral times for immunization subcutaneously through the back. Completefreund's adjuvant is used for the first time for immunization, whileincomplete freund's adjuvant is used to booster immunization.

Step 3: The blood samples are taken from the mice after the above immuneprocess, and the serum immune titer and immunosuppressive ability aredetected by indirect ELISA to select the mice with high cyproheptadineantibody content in serum.

Step 4: The selected mice were subjected to one last boosterimmunization with Incomplete Freund's adjuvant, and then, the impactimmunity is performed via intraperitoneal injection, usingcyproheptadine complete antigen without freund's adjuvant.

Step 5: The spleen cells and myeloma cells of BALB/c mice after impactimmunity are fused by the method of polyethylene glycol (PEG1500), andthe fusion cells were cultured in HAT medium. The positive cell poreswere detected by indirect-ELISA, and the inhibitory effect of positivecell pores was determined by indirect competitive ELISA. Subclones ofpositive cells with the best inhibition were performed by limiteddilution method, and the hybrid tumor cell lines that could secrete themonoclonal antibody of cyproheptadine were screened out.

Step 6: The sensitivity and specificity of the antibodies that wassecreted by hybrid tumor cell lines of secreting cytheptadine monoclonalantibodies was measured by ELISA.

In an embodiment of the invention, the molecular formula ofcyproheptadine hapten in step 1 is as follows:

In an embodiment of the invention, the interval between the firstimmunization and the booster immunization in steps 2 and 4 is one month,the interval between the booster immunization is 21 days, and theinterval between the booster immunization and the rush immunization is18 days.

In an embodiment of the invention, the immune process in steps 2 and 4comprises one time first immunization, three times booster immunizationand one-time rush immunization.

In an embodiment of the invention, the blood collection in step 3 isperformed on the seventh day after the end of the third immune process.

In one embodiment of the invention, the cell fusion in step 5 isperformed 3 days after the end of rush immunization.

In one embodiment of the invention, the above cyproheptadine hapten isprepared by steps of:

(1) the compound 1 was dissolved in methanol solution, and ethylchloroformate was added for stirring to obtain mixture.

(2) the cooled mixture in step (1) mentioned above was added HCL,extracted with ethyl acetate, the extracted organic layer wasconcentrated, and the compound 2 was obtained.

(3) the compound 2 was dissolved in ethyl alcohol and added potassiumhydroxide aqueous solution to stir to get mixture.

(4) the mixture in (3) was concentrated and extracted with ethylacetate, extracted with ethyl acetate, the extracted organic layer wasconcentrated, and the compound 3 was obtained.

(5) the compound 3 was dissolved in the mixture of Tetrahydrofuran THFsolution containing 3-chloropropionic acid, the triethylamine was addedstirring under N₂ to get mixture.

(6): the mixture in (5) was concentrated and extracted with ethylacetate, extracted with ethyl acetate, the extracted organic layer wasconcentrated, and the crude product of cyproheptadine haptens wasobtained.

The molecular formula of compound 1 is as follows:

The molecular formula of compound 2 is as follows:

The molecular formula of the compound 3 is as follows:

In a way of implementation of the invention, in (1), 5 g compound 1 ofconcentration of 17.4 mmol was dissolved in 50 mL methanol solution, and3 g ethyl chloroformate of concentration of 34.8 mmol was added forstirring under 120° C. for the night to obtain mixture.

In a way of implementation of the invention, in (2), the cooled mixturein (1) mentioned above was added HCL, extracted three times with 50 mLethyl acetate, the extracted organic layer was dried with anhydrousNa₂SO₄, after vacuum concentration the 4 g compound 2 was obtained.

In a way of implementation of the invention, in (3), 4 g compound 2 ofconcentration of 11.6 mmol was dissolved in 64 mL ethyl alcohol andadded dropwise potassium hydroxide aqueous solution of equivalent of 15to stir under 30° C. for the night to get mixture, which the aqueoussolution of potassium hydroxide is 10.3 g potassium hydroxide dissolvedin 16 mL H₂O to form a solution with a concentration of 174 mmol.

In a way of implementation of the invention, in (4), the mixture in step3 was extracted three times with 50 mL ethyl after acetatevacuumconcentration, and the extracted organic layer was dried with anhydrousNa₂SO₄, and the 2.5 g compound 3 was obtained after vacuumconcentration.

In a way of implementation of the invention, in (5), 2.5 g compound 3 ofthe concentration of 9.2 mmol was dissolved in the mixture of 30 mLtetrahydrofuran THF solution containing 1 g 3-chloropropionic acid ofthe concentration of 9.2 mmol, the 1 g triethylamine of theconcentration of 9.2 mmol was added stirring in 60° C. under for thenight N₂ to get mixture.

In a way of implementation of the invention, in (6), the mixture in (5)was 30 mL extracted three times with ethyl acetate after vacuumconcentration, the extracted organic layer was dried with anhydrousNa₂SO₄ and vacuum concentrated, and the crude product of cyproheptadinehaptens was obtained.

In a way of implementation of the invention, in (6), after purificationby silica gel chromatography (DCM/MeOH=20:1), 400 mg haptens wereobtained.

The invention provides application of cyproheptadine hapten in thepreparation cyproheptadine complete antigen, cyproheptadine antibody anda hybrid tumor cell line that secretes the monoclonal antibody ofcyproheptadine monoclonal antibody.

The invention provides a preparation method for the cyproheptadinecomplete antigen, which the cyproheptadine haptens was taken into brownflask adding MES buffer solution to dissolve, EDC and NHS was addedstirring to get the activated liquid; the keyhole limpet haemocyanin KLHwas dissolved in CB solution, the activated liquid was added adjustingpH and reacting. The cyproheptadine complete antigen was obtained afterisolating complete antigens and uncoupled small molecules.

The molecular formula of the cyproheptadine hapten is as follows:

The invention provides a preparation method for the cyproheptadinecomplete antigen, which 5 mg cyproheptadine haptens was taken into brownflask adding 1 mL MES buffer solution of pH4.7 and the concentration of0.1M to dissolve, 10 mg EDC and 6 mg NHS was added stirring for 6-8 h toget the activated liquid; the keyhole limpet haemocyanin KLH wasdissolved in CB solution, the activated liquid was added adjusting pH to9.0 with NaOH of the concentration of 1M, keeping in the dark andreacting for night. After isolating complete antigens and uncoupledsmall molecules, and identified by uv method, the cyproheptadinecomplete antigen was obtained.

The molecular formula of the cyproheptadine hapten is as follows:

The invention provides an application of cyproheptadine complete antigencharacterized by the use of the cyproheptadine complete antigen for thepreparation of cyproheptadine monoclonal antibodies.

The present invention provides a monoclonal antibody of cyproheptadine,which is obtained by secretion of a hybrid tumor cell line with thepreservation number CGMCC no. 14697.

The invention provides a preparation method of cyproheptadine monoclonalantibody, BALB/c mice were intraperitoneally injected with paraffin oil,and then intraperitoneally injected with hybrid tumor cells of secretingcyproheptadine monoclonal antibodies. After injection, ascites werecollected and purified, then the monoclonal antibody was preserved atlow temperature.

In another embodiment, BALB/c mice with 8 to 10 weeks, wereintraperitoneally injected with paraffin oil 1 mL each. After 7 days,each mouse intraperitoneally is injected with 1×106 hybrid tumor cells.From 7 days start collecting ascites, purified by bitter-ammoniumsulfate law, the monoclonal antibody was preserved at −20° C.

The present invention provides an application of cyproheptadinemonoclonal antibody in specifically identify cyproheptadine.

The present invention provides an application of a hybrid tumor cellline secreting cyproheptadine monoclonal antibody or the cyproheptadinehapten or the cyproheptadine complete antigen in obtaining detection kit

The advantages of the invention are:

The monoclonal antibody cell line obtained by the invention has a gooddetection sensitivity and specificity for cyproheptadine (IC50 value is0.37 ng/ml).

The invention provides a new synthetic method of cyproheptadine hapten,complete antigen and coating antigen.

The monoclonal antibody cell line obtained by the invention can be usedfor immunoassay detection.

Preserve Biological Materials

A hybrid tumor cell line that secretes monoclonal antibodies tocyproheptadine, which the classification is called monoclonal celllines, has been deposited with the general microbial center of the ChinaGeneral Microbial Culture Collection Committee (No. 3, yard 1, beichenwest road, chaoyang district, Beijing) under Accession Number CGMCC No.14697 on Sep. 5, 2017.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthetic process of cyproheptadine haptens.

FIG. 2 shows the UV characterization of cyproheptadine complete antigen

FIG. 3 shows the standard curve of inhibition of cyproheptadine bycyproheptadine monoclonal antibody.

DESCRIPTION OF PREFERRED EMBODIMENTS

The detailed implementation of the invention is further described asfollows. The following embodiments are used to illustrate the invention,but not to limit the scope of the invention.

Example 1

Synthesis of Cyproheptadine Hapten

5 g compound 1 of concentration of 17.4 mmol was dissolved in 50 mLmethanol solution, and 3 g ethyl chloroformate of concentration of 34.8mmol was added for stirring under 120° C. for the night to obtainmixture. the cooled mixture mentioned above was added HCL, extractedthree times with 50 mL ethyl acetate, the extracted organic layer wasdried with anhydrous Na₂SO₄, after vacuum concentration the 4 g compound2 was obtained. 4 g compound 2 of concentration of 11.6 mmol wasdissolved in 64 mL ethyl alcohol and added dropwise potassium hydroxideaqueous solution of equivalent of 15 to stir under 30° C. for the nightto get mixture, which the aqueous solution of potassium hydroxide is10.3 g potassium hydroxide dissolved in 16 mL H₂O to form a solutionwith a concentration of 174 mmol. the mixture was extracted three timeswith 50 mL ethyl after acetatevacuum concentration, and the extractedorganic layer was dried with anhydrous Na₂SO₄, and the 2.5 g compound 3was obtained after vacuum concentration. 2.5 g compound 3 of theconcentration of 9.2 mmol was dissolved in the mixture of 30 mLtetrahydrofuran THF solution containing 1 g 3-chloropropionic acid ofthe concentration of 9.2 mmol, the 1 g triethylamine of theconcentration of 9.2 mmol was added stirring in 60° C. under for thenight N₂ to get mixture. the mixture was 30 mL extracted three timeswith ethyl acetate after vacuum concentration, the extracted organiclayer was dried with anhydrous Na₂SO₄ and vacuum concentrated, and thecrude product of cyproheptadine haptens was obtained. After purificationby silica gel chromatography (DCM/MeOH=20:1), 400 mg haptens wereobtained.

Example 2

Synthesis of Cyproheptadine Complete Antigen

5 mg cyproheptadine haptens was taken into brown flask adding 1 mL MESbuffer solution of pH4.7 and the concentration of 0.1M to dissolve, 10mg EDC and 6 mg NHS was added stirring for 6-8 h to get the activatedliquid; the keyhole limpet haemocyanin KLH was dissolved in CB solution,the activated liquid was added adjusting pH to 9.0 with NaOH of theconcentration of 1M, keeping in the dark and reacting for night. Afterisolating complete antigens and uncoupled small molecules, andidentified by uv method, the cyproheptadine complete antigen wasobtained (the uv characterization effect of cyheptadine complete antigenof the present invention is shown in FIG. 2 ).

Example 3

Preparation of hybrid tumor cell lines that secrete the monoclonalantibody of cyproheptadine

Animal Immunization

After the cyproheptadine complete antigen and equivalent freund'sadjuvant were mixed and emulsified, BALB/c mice were immunized bysubcutaneous injection in the neck and back. For the first immunization,complete freund's adjuvant was used. For two times booster immunization,incomplete freund's adjuvant was used. The blood collection is performedon the seventh day after the end of the third immune process. Bloodsamples were taken from the mice after the above immune process, and theserum immune titer and immunosuppressive ability were detected byindirect ELISA to select the mice with high cyproheptadine antibodycontent in serum. The selected mice were subjected to 4th boosterimmunization with incomplete freund's adjuvant, and then, the rushimmunization is performed via intraperitoneal injection, usingcyproheptadine complete antigen without freund's adjuvant. (the intervalbetween the booster immunization is 21 days, and the interval betweenthe booster immunization and the rush immunization is 18 days).

2. Cell Fusion

After 3 days of shock immunity, cell fusion was performed by PEG(polyethylene glycol, with a molecular weight of 1500). The steps are asfollows:

The spleen of the mouse was taken aseptically, spleen cell suspensionwas obtained by grinding and screening with 200 mesh cells and thenCounting cells

Sp2/0 cells was collected, and suspended in rpm-1640 basic medium forcell counting.

Spleen cells and sp2/0 cells were mixed in a ratio of 1:10, centrifugedand fused with 50% PEG for 1 mi, the basic culture medium RPMI-1640 wasadded from slow to fast, after the centrifugal cells suspended inRPMI-1640 filter medium containing 20% fetal bovine serum and 2% of the50×HAT, and added to the 96 cell culture plate at 37° C. and cultivatingin 5% CO2 incubator.

Cell Screening and Cell Line Establishment

On the third day of cell fusion, the fusion cells were partiallyreplaced with the rpm-1640 screening medium, and on the fifth day, thecells were fully replaced with the rpm-1640 transition medium containing20% fetal bovine serum and 1% 100×HT, and the supernatant was taken onthe seventh day for screening.

Screening is divided into two steps: the first step was to screen outthe positive cells by indirect ELISA; in the second step, Melo wasselected as the standard product, and the inhibitory effect of positivecells was measured by indirect competitive ELISA. Cell pores that hadgood inhibition on all cyproheptadine standard products were selected,and subclone was conducted by finite dilution method. The same methodwas used for detection, and the cell lines were obtained after repeatedfor three times.

4. Cell Lines are Frozen

The hybrid tumor cell line named monoclonal cell line has been depositedwith the general microbial center of China General Microbial CultureCollection Committee (No. 3 yard 1 beichen west road chaoyang districtBeijing) under Accession Number CGMCC No. 14697 on Sep. 5, 2017.

Example 4 Preparation and Identification of Cyproheptadine MonoclonalAntibody

BALB/c mice with 8 to 10 weeks, were intraperitoneally injected withparaffin oil 1 mL each. After 7 days, each mouse intraperitoneally isinjected with 1×106 hybrid tumor cells secreting cyproheptadinemonoclonal antibody. From 7 days start collecting ascites, themonoclonal antibody was obtained after purification and preserved at−20° C.

Using indirect competition ELISA, the IC50 value of the cyproheptadinemonoclonal antibody was measured to be 0.37 ng/mL and the crossovervalue of the cyproheptadine analogue was measured to be less than 10%,which indicated that it has good sensitivity to cyproheptadine and couldbe used for cyproheptadine immunoassay.

Example 5

Application of Cyproheptadine Monoclonal Antibody

Monoclonal antibodies prepared by hybrid tumor cell lines were appliedto the addition and recovery test of ELISA about cyproheptadine. Thespecific steps are as follows:

The Solution Configuration

Carbonate buffer solution (CBS): Weigh Na₂CO₃ 1.59 g and NaHCO₃1.59 g,then mix with steaming water, plus double steamed water to 800 mL,adjust pH value to 9.6, add double steaming water for constant volume(1000 mL), store at 4° C.;

Phosphate Buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH₂PO₄,2.9 g Na₂HPO₄.12H₂O, dissolved in 800 mL pure water, pH was adjusted to7.2-7.4 with NaOH or HCl, then the capacity was constant to 1000 mL;

PBST: Add 0.5 ml Tween-20 to 1000 mL PBS solution (0.01 mol/L, pH 7.4);

Antibody Diluent: Washing buffer containing 0.1% gelatin;

TMB Substrate: Mixture of solution A and solution B with 1:5; SolutionA: Na₂HPO₄.12H₂O 18.43 g, citric acid 9.33 g, constant to 1000 mL withpure water. Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol.

(2) Coating: coating antigen CLA-CMO-OVA had reacted for 2 h afterserial dilution of the carbonate buffer (pH 9.6, 0.05 m) from 1 μg/mL,100 μL/hole at 37° C.

(3) Washing: the solution in the plate was poured out and wash it 3times with wash solution, 3 min each.

(4) Sealing: After pat dry, 200 μL/hole sealing fluid was added toreaction within 2 h at 37° C., drying after washing.

(5) Sample adding: antiserum was diluted from the ratio of 1:1000, andwas added into the degree of the dilution of coating hole, 100 μL/hole,and had reacted at 70° C. for 30 min. After fully washing, HRP—Goat antiMouse IgG that had diluted with the ratio of 1:3000 was added andreacted at 37° C. for 30 min, 100 μL/hole;

(6) Coloration: The enzyme label plate was taken out, after washing,each hole was added into TMB Color liquid, and reacted at 37° C. for 15min avoiding light.

(7) Termination and determination: To terminate the reaction, 50 μLtermination solution was added to each hole, and then the value of OD₄₅₀of each hole was measured with an enzyme marker.

(8) Reading result: The ELISA titer of serum was the highest dilutionfactor corresponding to the serum with the OD₄₅₀ value greater than orequal to 2.1 times of the negative control hole (i.e., P/N≥2.1).

The IC50 of the monoclonal antibody was 0.37 ng/mL, indicating a goodsensitivity to cyproheptadine and can be used for cyproheptadineimmunoassay.

Serum Test Results of Mice after Three Immunizations:

The concentra- The dilution tion of standard ratio of The concentrationof coating antigen ng/ml mice serum 1 μg/ml 0.3 μg/ml 0.1 μg/ml  0 ng/ml1k 2.983 2.623 1.278 3k 2.618 2.134 0.844 9k 1.973 1.328 1.497 20 ng/ml1k 2.163 1.28 0.419 3k 1.597 0.751 0.248 9k 0.746 0.338 0.136 OD OD(Standard addition Small molecule screening Cell line (withoutconcentration to be measured process number standard) Xng/ml)cyproheptadine After the 4A11 1.768 0.282(5 ng/ml)  fusion 1 g 2H121.338 0.078(2 ng/ml)  2 g 4B1 1.244 0.468(0.5 ng/ml) 3 g 3E′ 10 1.3790.612(0.5 ng/ml)

Contrast Example 1: Preparation of Hybrid Tumor Cell Lines SecretingCyproheptadine Monoclonal Antibody

Replacing the coating antige in the above embodiments with BSA, it wasfound that cyproheptadine conjugated OVA was more effective as thecoating antigen.

The concentra- The concentra- The coating tion of tion of IC50 antigencoating antibody ODmax (ng/ml) CHP-OVA 40:1 0.3 μg/ml 0.3 μg/ml 1.4570.37 CHP-BSA 60:1 0.3 μg/ml 0.3 μg/ml 1.895 0.514

Contrast Example 2: Preparation of Hybrid Tumor Cell Lines SecretingCyproheptadine Monoclonal Antibody

In the above embodiment, the number of addition and exemption waschanged to the serum test results of mice after the Secondaryimmunization and exemption:

The concentra- The dilution tion of standard ratio of The concentrationof coating antigen ng/ml mice serum 1 μg/ml 0.3 μg/ml 0.1 μg/ml  0 ng/ml1k 2.683 2.323 0.978 3k 2.318 1.834 0.544 9k 1.673 1.028 0.297 20 ng/ml1k 2.141 1.318 0.519 3k 1.647 0.815 0.359 9k 0.864 0.479 0.018

Compared with the three times booster immunizations, the titer andinhibition of serum after the two times booster immunizations were notas good as those after the three times booster immunizations.

The above description is only a preferred method of implementation ofthe invention and is not used to limit the invention. It should be notedthat, for ordinary technical personnel in the field of technology, someimprovements and variations can be made under the technical principlesof the invention. These improvements and variations should also beconsidered as the scope of protection of the invention.

What is claimed is:
 1. A hybridoma cell line that secretescyproheptadine monoclonal antibodies deposited with the generalmicrobial center of the China General Microbial Culture CollectionCommittee (No. 3, yard 1, beichen west road, chaoyang district, Beijing)under Accession Number CGMCC No. 14697 on Sep. 5,
 2017. 2. Acyproheptadine monoclonal antibody obtained through the hybridoma cellline that secretes cyproheptadine monoclonal antibodies according toclaim
 1. 3. The cyproheptadine monoclonal antibody according to claim 2wherein the antibody is applicable in specifically identifyingcyproheptadine.